Project Summary In addition to well-documented G protein?dependent and ?-arrestin-dependent GPCR signaling pathways, other cellular effectors are recruited to GPCRs. Signal adaptor protein 14-3-3 is one of such cellular effectors engaged with GPCRs. 14-3-3 proteins are ubiquitously expressed in cells, but their highest expression is found in the brain. Although biochemical evidence shows 14-3-3 forms complexes with some GPCRs, investigation of GPCR-mediated 14-3-3 signaling has been largely ignored. Lack of ability to assess specific 14-3-3 signaling is a major reason for studies to lag behind studies of G-protein and ?-arrestin signaling pathways. We have developed a new assay for assessing GPCR-mediated 14-3-3 signaling by measuring GPCR and 14- 3-3 interactions in the phase 1 study. We demonstrate that GPCR-mediated 14-3-3 signaling is ligand- regulated. Multiple GPCRs interact with 14-3-3 proteins in response to agonist stimulation. GPCR-mediated 14-3-3 signaling is phosphorylation-dependent, and GPCR/14-3-3 interaction likely takes place after receptor desensitization and internalization. GPCR-mediated 14-3-3 signaling can be ?-arrestin-independent and agonists can have different potencies in 14-3-3 and ?-arrestin signaling pathways. GPCRs can also mediate 14-3-3 and Raf-1 kinase interaction. Our work opens up a new broad realm of previously unappreciated GPCR signal transduction. GPCR/14-3-3 LinkLight assay cells offer novel tools for GPCR drug discovery. It is likely that GPCR-mediated 14-3-3 signaling is a more general phenomenon than we have previously realized. In the Phase II research plan, we will continue characterizing and developing a large number of commercial GPCR/14-3-3 assay cell lines for brain-derived GPCRs including serotonin, dopamine, opioid, orexin, somatostatin, muscarinic, cannabinoid, adrenergic, and neuropeptide receptors. These receptors- mediated 14-3-3 signaling pathway has yet to be characterized individually. These GPCR/14-3-3 cell lines would be novel assay tools to aid us to study GPCR signaling and to develop new drugs. We will also investigate the potential that metabolic glutamate receptors (GPCR family C members) interact with 14-3-3 proteins. Metabolic glutamate receptors (GRMs) can signal through G-proteins, but they do not recruit ?-arrestins and have no GPCR-mediated ?-arrestin signaling. We have showed that GPCR-mediated 14-3-3 signaling pathway can be ?-arrestin-independent by using ADRB3 as an example (completed Phase 1 extra task 3). Thus, if we can demonstrate that metabolic glutamate receptors can mediate 14-3-3 signaling, we will have a new approach to target these important receptors. Finding a biased 14-3-3 signaling ligand would be another important discovery. We will collaborate with Professor Thomas Sakmar in Rockefeller University for a pilot screen. Prof. Sakmar has been studying the human dopamine D4 receptor (hDRD4) for which there are three exon variants in the human populations (D4.2, D4.4 and D4.7). We will screen for biased ligands that have differential signaling between 14-3-3 and ?- arrestin signaling pathways by using theD4.4 variant as the model. If we find biased hits in the screen, we will cross check the hits with different variants. Biased ligands would be valuable probes for characterizing the physiological significance of GPCR-mediated 14-3-3 signaling.